Nonfixable Viability Dyes for Flow Cytometry

Dead cells often give false positive results, as they tend to bind nonspecifically to many reagents. Therefore, removing dead cells from your flow cytometry data is a critical step to help ensure accurate results and analysis. Molecular Probes® nonfixable cell viability assays for flow cytometry offer researchers options for distinguishing live and dead cell populations that are more accurate than forward- and side-scatter data.

Viability assays using cell-impermeant SYTOX® DNA-binding dyes

SYTOX® Dead Cell Stains do not cross intact cell membranes, and they exhibit increased fluorescence upon dsDNA binding, making them some of our most brilliant dead cell stains (Figure 1). Easy to use, SYTOX® Dead Cell Stains can be applied to cells and visualized without an additional wash step because they are nonfluorescent in aqueous media. Available in multiple single-color formats and compatible with multiple lasers, these dyes provide the flexibility you need. These stains can be used on multiple platforms, including fluorescence microscopy, flow cytometry, and microplates.

Viability assays using classic cell-impermeant DNA-binding dyes

Cell-impermeant classic DNA-binding dyes include propidium iodide (Figure 2) and 7-AAD. Both of these dyes have been used extensively for viability assays in flow cytometry, since they can be excited by the 488 or 532 nm laser and emit at wavelengths beyond 610 nm.

Viability assays using esterase substrates

CellTrace™ Calcein AM dyes can be passively loaded into adherent and nonadherent cells. These cell-permeant esterase substrates serve as viability probes that measure both enzymatic activity, which is required to activate their fluorescence, and cell membrane integrity, which is required for intracellular retention of their fluorescent products. Available with blue, violet, and green fluorescence, these dyes are ideal for short-term staining of live cells and can be used in multiplexed flow cytometry experiments (Figure 3).

Figure 3. CellTrace™ Calcein Green AM staining of live cells. A 1:1 mixture of live and ethanol-fixed (dead) human B cells was stained with calcein green AM and ethidium homodimer-1. After 5 minutes, flow cytometric analysis was carried out with excitation at 488 nm. The resulting bivariate frequency distribution shows the clear separation of the green-fluorescent (530 nm) live cell population from the red-fluorescent (585 nm) dead cell population.

Viability assays using esterase substrates and amine-reactive dyes

LIVE/DEAD® Violet Viability/Vitality Kit

The LIVE/DEAD® Violet Viability/Vitality Kit provides a two-color fluorescence cell viability and vitality assay based on the simultaneous determination of live and dead cells with two probes that measure recognized parameters of cell health: plasma membrane integrity as a measure of cell viability, and intracellular esterase activity as a measure of cell vitality (Figure 4). CellTrace™ Calcein Violet AM and LIVE/DEAD® Fixable Aqua fluorescent reactive dye are optimal dyes for this application; both dyes utilize the violet laser, allowing other laser lines to be used with more conventional markers.

Figure 4. Staining of a mixture of heat-killed and untreated Jurkat cells. Jurkat cells were stained according to the protocol in the LIVE/DEAD® Violet Viability/Vitality Kit. Cells were analyzed using a flow cytometer equipped with a 405 nm laser and a 450/50 nm bandpass filter for calcein violet–labeled live cells (L), and a 525/50 nm bandpass filter for the aqua dye–labeled dead cells (D).

SYTOX® Dead Cell Stains enter cells with compromised cell membranes and bind to dsDNA/RNA. The dyes are mostly nonfluorescent until they bind to nucleic acids. Cannot pass through intact cell membranes and are not fixable. May also be used for DNA content cell cycle analysis in fixed cells but does require the addition of RNase. Allows exclusion of dead cells from flow cytometric analysis.

Classic DNA binding dead cell dyes enter cells with compromised cell membranes and bind to dsDNA/RNA. Cannot pass through intact cell membranes and are not fixable. May also be used for DNA content cell cycle analysis in fixed cells but this requires the addition of RNase. Allows exclusion of dead cells from flow cytometric analysis.

CellTrace™ calcein AM dyes are nonfluorescent when the AM ester is intact and they cross membranes of all cells. Once inside live cells, the active esterases cleave the AM group which allows the dye to fluoresce. The AM group is not cleaved in dead cells and the dyes do not fluoresce. Best use is with short-term labeling as the dye, once the AM group is cleaved, can be actively transported out of the cell within a few hours.

The LIVE/DEAD® Violet Viability/Vitality Kit provides a two-color fluorescence cell viability and vitality assay that is based on the simultaneous determination of live and dead cells with two probes that measure recognized parameters of cell health: plasma membrane integrity as a measure of cell viability, and intracellular esterase activity as a measure of cell vitality.

Fluorescent label

Calcein Violet, AM

LIVE/DEAD® Aqua stain

Readout

Live cells are highly fluorescent; dead cells are significantly dimmer

Live cells are minimally fluorescent; dead cells are highly fluorescent